Philip C. Schouten

REV7 counteracts DNA double-strand break resection and affects PARP inhibition

Error-free repair of DNA double-strand breaks (DSBs) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway. In the absence of BRCA1-mediated HR, the administration of PARP inhibitors induces synthetic lethality of tumour cells of patients with breast or ovarian cancers. Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration. Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases. In particular, little is known about BRCA1-independent restoration of HR. Here we show that loss of REV7 (also known as MAD2L2) in mouse and human cell lines re-establishes CTIP-dependent end resection of DSBs in BRCA1-deficient cells, leading to HR restoration and PARP inhibitor resistance, which is reversed by ATM kinase inhibition. REV7 is recruited to DSBs in a manner dependent on the H2AX–MDC1–RNF8–RNF168–53BP1 chromatin pathway, and seems to block HR and promote end joining in addition to its regulatory role in DNA damage tolerance. Finally, we establish that REV7 blocks DSB resection to promote non-homologous end-joining during immunoglobulin class switch recombination. Our results reveal an unexpected crucial function of REV7 downstream of 53BP1 in coordinating pathological DSB repair pathway choices in BRCA1-deficient cells.

Integration of genomic, transcriptomic and proteomic data identifies two biologically distinct subtypes of invasive lobular breast cancer

Invasive lobular carcinoma (ILC) is the second most frequently occurring histological breast cancer subtype after invasive ductal carcinoma (IDC), accounting for around 10% of all breast cancers. The molecular processes that drive the development of ILC are still largely unknown. We have performed a comprehensive genomic, transcriptomic and proteomic analysis of a large ILC patient cohort and present here an integrated molecular portrait of ILC. Mutations in CDH1 and in the PI3K pathway are the most frequent molecular alterations in ILC. We identified two main subtypes of ILCs: (i) an immune related subtype with mRNA up-regulation of PD-L1, PD-1 and CTLA-4 and greater sensitivity to DNA-damaging agents in representative cell line models; (ii) a hormone related subtype, associated with Epithelial to Mesenchymal Transition (EMT), and gain of chromosomes 1q and 8q and loss of chromosome 11q. Using the somatic mutation rate and eIF4B protein level, we identified three groups with different clinical outcomes, including a group with extremely good prognosis. We provide a comprehensive overview of the molecular alterations driving ILC and have explored links with therapy response. This molecular characterization may help to tailor treatment of ILC through the application of specific targeted, chemo- and/or immune-therapies.

Impact of Intertumoral Heterogeneity on Predicting Chemotherapy Response of BRCA1-Deficient Mammary Tumors

The lack of markers to predict chemotherapy responses in patients poses a major handicap in cancer treatment. We searched for gene expression patterns that correlate with docetaxel or cisplatin response in a mouse model for breast cancer associated with BRCA1 deficiency. Array-based expression profiling did not identify a single marker gene predicting docetaxel response, despite an increase in Abcb1 (P-glycoprotein) expression that was sufficient to explain resistance in several poor responders. Intertumoral heterogeneity explained the inability to identify a predictive gene expression signature for docetaxel. To address this problem, we used a novel algorithm designed to detect differential gene expression in a subgroup of the poor responders that could identify tumors with increased Abcb1 transcript levels. In contrast, standard analytical tools, such as significance analysis of microarrays, detected a marker only if it correlated with response in a substantial fraction of tumors. For example, low expression of the Xist gene correlated with cisplatin hypersensitivity in most tumors, and it also predicted long recurrence-free survival of HER2-negative, stage III breast cancer patients treated with intensive platinum-based chemotherapy. Our findings may prove useful for selecting patients with high-risk breast cancer who could benefit from platinum-based therapy.

BRCA1-like signature in triple negative breast cancer : Molecular and clinical characterization reveals subgroups with therapeutic potential

Triple negative (TN) breast cancers make up some 15% of all breast cancers. Approximately 10–15% are mutant for the tumor suppressor, BRCA1. BRCA1 is required for homologous recombination‐mediated DNA repair and deficiency results in genomic instability. BRCA1‐mutated tumors have a specific pattern of genomic copy number aberrations that can be used to classify tumors as BRCA1‐like or non‐BRCA1‐like. BRCA1 mutation, promoter methylation, BRCA1‐like status and genome‐wide expression data was determined for 112 TN breast cancer samples with long‐term follow‐up. Mutation status for 21 known DNA repair genes and PIK3CA was assessed. Gene expression and mutation frequency in BRCA1‐like and non‐BRCA1‐like tumors were compared. Multivariate survival analysis was performed using the Cox proportional hazards model. BRCA1 germline mutation was identified in 10% of patients and 15% of tumors were BRCA1 promoter methylated. Fifty‐five percent of tumors classified as BRCA1‐like. The functions of genes significantly up‐regulated in BRCA1‐like tumors included cell cycle and DNA recombination and repair. TP53 was found to be frequently mutated in BRCA1‐like (P < 0.05), while PIK3CA was frequently mutated in non‐BRCA1‐like tumors (P < 0.05). A significant association with worse prognosis was evident for patients with BRCA1‐like tumors (adjusted HR = 3.32, 95% CI = 1.30–8.48, P = 0.01). TN tumors can be further divided into two major subgroups, BRCA1‐like and non‐BRCA1‐like with different mutation and expression patterns and prognoses. Based on these molecular patterns, subgroups may be more sensitive to specific targeted agents such as PI3K or PARP inhibitors.

Breast cancer in women at high risk: the role of rapid genetic testing for BRCA1 and -2 mutations and the consequences for treatment strategies.

Specific clinical questions rise when patients, who are diagnosed with breast cancer, are at risk of carrying a mutation in BRCA1 and -2 gene due to a strong family history or young age at diagnosis. These questions concern topics such as 1. Timing of genetic counseling and testing, 2. Choices to be made for BRCA1 or -2 mutation carriers in local treatment, contralateral treatment, (neo)adjuvant systemic therapy, and 3. The psychological effects of rapid testing. The knowledge of the genetic status might have several advantages for the patient in treatment planning, such as the choice whether or not to undergo mastectomy and/or prophylactic contralateral mastectomy. The increased risk of developing a second breast cancer in the ipsilateral breast in mutation carriers, is only slightly higher after primary cancer treatment, than in the general population. Prophylactic contralateral mastectomy provides a substantial reduction of contralateral breast cancer, although only a small breast cancer specific survival benefit. Patients should be enrolled in clinical trials to investigate (neo)-adjuvant drug regimens, that based on preclinical and early clinical evidence might be targeting the homologous recombination defect, such as platinum compounds and PARP inhibitors. If rapid testing is performed, the patient can make a well-balanced decision. Although rapid genetic counseling and testing might cause some distress, most women reported this approach to be worthwhile. In this review the literature regarding these topics is evaluated. Answers and suggestions, useful in clinical practice are discussed.

Breast Cancers with a BRCA1-like DNA Copy Number Profile Recur Less Often Than Expected after High-Dose Alkylating Chemotherapy

Purpose: Breast cancers in carriers of inactivating mutations of the BRCA1 gene carry a specific DNA copy-number signature (“BRCA1-like”). This signature is shared with cancers that inactivate BRCA1 through other mechanisms. Because BRCA1 is important in repair of DNA double-strand breaks through error-free homologous recombination, patients with a BRCA1-like tumor may benefit from high-dose alkylating (HD) chemotherapy, which induces DNA double-strand breaks. Experimental Design: We investigated a single institution cohort of high-risk patients that received tandem HD chemotherapy schedule comprising ifosfamide, epirubicin, and carboplatin or conventional chemotherapy. We classified copy-number profiles to be BRCA1-like or non–BRCA1-like and analyzed clinical associations and performed survival analysis with a treatment by biomarker interaction design. Results: BRCA1-like status associated with high-grade and triple-negative breast cancers. BRCA1-like cases benefitted from the HD compared with a conventional regimen on disease-free survival (DFS): [hazard ratio (HR), 0.05; 95% confidence interval (CI), 0.01–0.38; P = 0.003]; distant DFS (DDFS): (HR, 0.06; 95% CI, 0.01–0.43; P = 0.01); and overall survival (OS; HR, 0.15; 95% CI, 0.03–0.83; P = 0.03) after correction for prognostic factors. No such benefit was observed in the non–BRCA1-like cases on DFS (HR, 0.74; 95% CI, 0.38–1.46; P = 0.39), DDFS (HR, 0.79; 95% CI, 0.41–1.52; P = 0.47), and OS (HR, 0.93; 95% CI, 0.52–1.64; P = 0.79). The P values for interaction were 0.01 (DFS), 0.01 (DDFS), and 0.045 (OS). Conclusions: BRCA1-like tumors recurred significantly less often after HD than conventional chemotherapy. BRCA1-like copy-number profile classification may be a predictive marker for HD alkylating chemotherapy. Clin Cancer Res; 21(4); 763–70. ©2014 AACR.

Platform comparisons for identification of breast cancers with a BRCA-like copy number profile

Previously, we employed bacterial artificial chromosome (BAC) array comparative genomic hybridization (aCGH) profiles from BRCA1 and -2 mutation carriers and sporadic tumours to construct classifiers that identify tumour samples most likely to harbour BRCA1 and -2 mutations, designated ‘BRCA1 and -2-like’ tumours, respectively. The classifiers are used in clinical genetics to evaluate unclassified variants, and patients for which no good quality germline DNA is available. Furthermore, we have shown that breast cancer patients with BRCA-like tumour aCGH profiles benefit substantially from platinum-based chemotherapy, potentially due to their inability to repair DNA double strand breaks (DSB), providing a further important clinical application for the classifiers. The BAC array technology has been replaced with oligonucleotide arrays. To continue clinical use of existing classifiers, we mapped oligonucleotide aCGH data to the BAC domain, such that the oligonucleotide profiles can be employed as in the BAC classifier. We demonstrate that segmented profiles derived from oligonucleotide aCGH show high correlation with BAC aCGH profiles. Furthermore, we trained a support vector machine score to objectify aCGH profile quality. Using the mapped oligonucleotide aCGH data, we show equivalence in classification of biologically relevant cases between BAC and oligonucleotide data. Furthermore, the predicted benefit of DSB inducing chemotherapy due to a homologous recombination defect is retained. We conclude that oligonucleotide aCGH data can be mapped to and used in the previously developed and validated BAC aCGH classifiers. Our findings suggest that it is possible to map copy number data from any other technology in a similar way.

High XIST and Low 53BP1 Expression Predict Poor Outcome after High-Dose Alkylating Chemotherapy in Patients with a BRCA1-like Breast Cancer

In previous studies, high expression of XIST and low expression of 53BP1 were respectively associated with poor systemic therapy outcome in patients and therapy resistance in BRCA1-deficient mouse tumor models, but have not been evaluated in BRCA1-deficient patients. Previously, we demonstrated that classifying breast cancer copy number profiles as BRCA1-like or non–BRCA1-like identified patients enriched for defects in BRCA1 that benefit from high-dose (HD) alkylating chemotherapy compared with a conventional standard regimen. We investigated whether XIST and 53BP1 expression predicted poor outcome of HD chemotherapy within 28 BRCA1-like patients from a trial randomizing between HD [4 cycles 5-fluorouracil, epirubicin, cyclophosphamide (FEC) followed by 1 cycle HD carboplatin, thiotepa, cyclophosphamide] or conventional chemotherapy (5 cycles FEC), for which both XIST and 53BP1 statuses were available. High RNA expression of XIST (n = 5) and low protein expression of 53BP1 (n = 3) expression did not coincide. Patients with either one had poor outcome after treatment with HD chemotherapy, whereas patients with low expression of XIST and high expression of 53BP1 derived substantial benefit of this regimen on recurrence-free survival, disease-free survival, and overall survival, corroborating preclinical findings. XIST and 53BP1 may be predictive biomarkers in BRCA1-like breast cancer. Mol Cancer Ther; 15(1); 190–8. ©2015 AACR.

Challenges in the Use of DNA Repair Deficiency As a Biomarker in Breast Cancer

The development of drug-biomarker combinations holds promise for tailoring treatment for patients with cancer on the basis of the biology of the tumor exemplified by estrogen receptor expression and blockade and human epidermal growth factor receptor 2 targeting. Approximately 85% of breast cancers have a target for therapy, but triple-negative breast cancers (TNBCs) do not. Although trials have focused on identifying the most efficacious anticancer drug (regimen) for this subtype, it may be that predictive biomarkers are required to guide treatment choices because of the intrinsic heterogeneity of TNBCs. Molecular characterizations have demonstrated a strong association between TNBCs and BRCA1 mutations. Preclinical mechanistic insight indicated that tumor cells with a defect in BRCA1 have impaired homologous recombination (HR), the only error-free pathway of repair of interstrand crosslinks. Cells with impaired HR display sensitivity to agents that induce such lesions. In the general breast cancer population, platinum agents that cause interstrand crosslinks are not preferred over other treatment regimens. However, given the possibility that TNBCs are enriched for such a targetable HR deficiency (HRD), platinum has attracted renewed interest as has been shown in recently published articles. Furthermore, TNBCs may be enriched for BRCA1 mutations and also for a larger group of nonmutated tumors that exhibit HRD. The use of platinum in TNBCs with biomarker analyses has been investigated in the two articles that accompany this editorial. Telli et al neoadjuvantly treated patients with triple-negative or BRCA1/ 2-associated breast cancer with a regimen containing gemcitabine, carboplatin, and iniparib in the PrECOG 0105 trial. Overall, a pathologic complete remission rate of 36% and acceptable toxicity of the regimen were reported. Isakoff et al conducted a phase II trial with cisplatin or carboplatin in patients with metastatic TNBC (TBCRC009; Platinum for Triple-Negative Metastatic Breast Cancer and Evaluation of p63/p73 as a Biomarker of Response). The overall response rate was 25%. Both studies reproduce previous findings of efficacy of a platinum-based regimen or platinum alone in TNBC. However, only a subset of patients in these trials derived clinical benefit. Therefore, secondary investigations may help identify biomarkers that associate with a preferential benefit from use of a platinum regimen, which may help guide therapy decisions. Both studies report their analysis of BRCA germline mutation status as well as potential biomarkers for assessing HRD, specifically the HRD loss of heterozygosity (HRD-LOH) score and the HRD large-scale transition (HRD-LST) score. Both markers evaluate so-called genomic scars as signatures of aberrations that can be observed from singlenucleotide polymorphism array–based sequencing or similar technologies and that are associated with defects in error-free repair of interstrand crosslinks. The HRD-LOH score counts the numbers of LOH regions larger than 15 MB but smaller than chromosome size, whereas the HRD-LST score counts the number of breaks between adjacent segments of at least 10 MB. Cutoffs were trained to best separate tumors that had a known deleterious aberration in BRCA1/2 from those that did not. In both studies, patients with a BRCA germline mutation had higher response rates than the general cohort, and the HRD scores highly correlated with BRCA1/2 germline mutations and response rates. In addition, Telli et al identified BRCA1 methylation in nonmutated tumors with high HRD-LOH score, which suggests that these scores identify a subgroup of patients who could benefit from therapy that targets their HRD. Unfortunately, single-arm phase II studies do not allow proper evaluation of a biomarker as a predictor of response to therapy, and they offer limited insight on how to exploit the observed association with outcome of BRCA-associated biomarkers (Fig 1). Thus, the studies by Telli et al and Isakoff et al join several other articles published over the last decade that have reported on the potential promise of BRCA-associated HRD biomarkers but did not provide definitive data on clinical utility. It has been established that the optimal biomarker trial design requires biomarker-positive and -negative patients and patients who have been treated with and without the therapy of interest to evaluate both prognostic and predictive signals. If not included in the phase II design, such a control group could be added. Hence, the lack of a control arm in these two new studies is indeed a major limitation of their findings. In contrast, the TNT trial (Triple Negative Breast Cancer Trial, recently reported as an abstract) was able to more appropriately evaluate the use of HRD biomarkers, at least in patients with advanced disease. In that trial, patients with locally advanced or metastatic TNBC were randomly assigned to therapy with carboplatin at area under the [concentration-time] curve 6 or standardof-care docetaxel. Of great interest, among the 376 patients in that study, 29 were known to be BRCA1 or BRCA2 mutation carriers (including 13 who had estrogen receptor–positive, HER2-negative disease). Prespecified analyses specifically addressed populations JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 33 NUMBER 17 JUNE 1

Lack of Genomic Heterogeneity at High-Resolution aCGH between Primary Breast Cancers and Their Paired Lymph Node Metastases

Lymph-node metastasis (LNM) predict high recurrence rates in breast cancer patients. Systemic treatment aims to eliminate (micro)metastatic cells. However decisions regarding systemic treatment depend largely on clinical and molecular characteristics of primary tumours. It remains, however, unclear to what extent metastases resemble the cognate primary breast tumours, especially on a genomic level, and as such will be eradicated by the systemic therapy chosen. In this study we used high-resolution aCGH to investigate DNA copy number differences between primary breast cancers and their paired LNMs. To date, no recurrent LNM-specific genomic aberrations have been identified using array comparative genomic hybridization (aCGH) analysis. In our study we employ a high-resolution platform and we stratify on different breast cancer subtypes, both aspects that might have underpowered previously performed studies.To test the possibility that genomic instability in triple-negative breast cancers (TNBCs) might cause increased random and potentially also recurrent copy number aberrations (CNAs) in their LNMs, we studied 10 primary TNBC–LNM pairs and 10 ER-positive (ER+) pairs and verified our findings adding additionally 5 TNBC-LNM and 22 ER+-LNM pairs. We found that all LNMs clustered nearest to their matched tumour except for two cases, of which one was due to the presence of two distinct histological components in one tumour. We found no significantly altered CNAs between tumour and their LNMs in the entire group or in the subgroups. Within the TNBC subgroup, no absolute increase in CNAs was found in the LNMs compared to their primary tumours, suggesting that increased genomic instability does not lead to more CNAs in LNMs. Our findings suggest a high clonal relationship between primary breast tumours and its LNMs, at least prior to treatment, and support the use of primary tumour characteristics to guide adjuvant systemic chemotherapy in breast cancer patients.